OsNAR2.1 Interaction with OsNIT1 and OsNIT2 Functions in Root-growth Responses to Nitrate and Ammonium.

The nitrate transport accessory protein OsNAR2 plays a critical role in root-growth responses to nitrate and nitrate acquisition in rice (). In this study, a pull-down assay combined with yeast two-hybrid and coimmunoprecipitation analyses revealed that OsNAR2.1 interacts with OsNIT1 and OsNIT2. Moreover, an in vitro nitrilase activity assay indicated that indole-3-acetonitrile (IAN) is hydrolyzed to indole-3-acetic acid (IAA) by OsNIT1, the activity of which was enhanced 3- to 4-fold by OsNIT2 and in excess of 5- to 8-fold by OsNAR2.1. Knockout (KO) of was accompanied by repressed expression of both and , whereas KO of and in the and mutant lines did not affect expression of or the root nitrate acquisition rate. and displayed decreased primary root length and lateral root density. Double KO of and caused further decreases in lateral root density under nitrate supply. Ammonium supply repressed expression whereas it upregulated and expression. Both and showed root growth hypersensitivity to external ammonium; however, less root growth sensitivity to external IAN, higher expression of three IAA-amido synthetase genes, and a lower rate of H-IAA movement toward the roots were observed. Taken together, we conclude that the interaction of OsNIT1 and OsNIT2 activated by OsNAR2.1 and nitrogen supply is essential for maintaining root growth possibly via altering the IAA ratio of free to conjugate forms and facilitating its transportation.